Compound specific isotope analysis was combined with phospholipid fatty acid (PLFA) analysis to identify methanotrophic activity in members of the sedimentary microbial community in the Altamaha and Savannah River estuaries in Georgia. 13C-depleted PLFAs indicate methane utilization because methane is the only C source with a depleted isotopic signal (-45 to -72 per mil) in these ecosystems. Three PLFAs (16:1w7, 16:1w5, and 10Me16:0) had 13C-depleted values (-52.37 to -39.2 per mil) during winter and spring when the salinity was less than 5 ppt. No depleted signal was observed during summer when the salinity was higher (4 to 30 ppt). 10Me16:0, an indicator of sulfate reducing bacteria (SRB), showed 13C-depleted values only in the Altamaha, indicating a discrepancy in the activity of specific functional groups depending on location. Methane production was higher in the Altamaha River than in the Savannah and its production did not correlate with the detection of 13C-depleted PLFAs. Our results suggest that methane oxidation occurs on a seasonal basis in highly dynamic estuaries and is the result of more than one microbial functional group (methanotrophs and SRB) in shallow estuarine sediments.